Fig. 10
From: Munc18-1 is crucial to overcome the inhibition of synaptic vesicle fusion by αSNAP

αSNAP potently inhibits fusion mediated by pre-formed trans-SNARE complexes. a Diagram illustrating the experimental design. The assays monitored the development of FRET between V-liposomes containing Alex488-synaptobrevin and T-liposomes containing tetramethylrhodamine (TMR)-syntaxin-1 and WT SNAP-25 upon trans-SNARE complex assembly. The design is analogous to that of Fig. 6a, but the use of WT SNAP-25 allows liposome fusion. The V-liposomes contained DiD-labeled lipids to monitor lipid mixing from de-quenching of the DiD fluorescence. V- and T-liposome samples were incubated with Syb49–93 for 24 h at 4 °C to promote trans-SNARE complex assembly without liposome fusion, and the temperature was raised to 37 °C after adding various factors to allow lipid mixing. b Fluorescence emission spectra (468 nm excitation) of a mixture of V-liposomes containing Alexa-488-synaptobrevin and T-liposomes containing TMR-syntaxin-1 and WT SNAP-25 (1:4 V- to T-liposome ratio) that had been incubated for 24 h with Syb49–93 at 4 °C (red trace) and of the same sample after adding 2 µM αSNAP (blue trace) or 2 µM αSNAP plus 0.4 µM NSF, 2 mM ATP, and 2.5 mM Mg2+ (orange trace). The black curve shows a control spectrum obtained by adding spectra acquired separately for V- and T-liposomes. c–f Lipid mixing assays performed as summarized in a. De-quenching of DiD fluorescence was monitored after adding the indicated reagents to the trans-SNARE complexes pre-formed at 4 °C and raising the temperature to 37 °C. c Assays performed with no additions (red trace) or adding 2 µM αSNAP without (blue trace) or with 0.4 µM NSF (orange trace). The black trace shows an experiment were the V- and T-liposomes were mixed at 37 °C without pre-incubation at 4 °C. d Assays analogous to those in b with the addition of 2 µM WT αSNAP or the αSNAP FS or KE mutants. e Assays analogous to those of c with the addition of different concentrations of αSNAP. f Assays analogous to those of c with the addition of different combinations of 1 µM Munc18-1 (M18), 0.3 µM M13C1C2BMUNC2C (M13), 0.4 µM NSF, 2 µM αSNAP, and 0.5 mM Ca2+. Source data are provided as a Source Data file